The rodent incisor provides an excellent model system for studying stem cells because it grows continuously and generates all the necessary cell types from an active pool of adult stem cells. Using mouse genetics, scientists have begun to understand how these cells are regulated by different signaling pathways. However, the exact mechanism that controls their proliferation and differentiation requires further studies. In particular, in light of recent in vitro experiments showing that stem cell behavior can be modified by mechanical force, it is plausible that incisor stem cells are similarly regulated by their physial environment, an area that is currently poorly understood. Objective/hypothesis: The Hippo signaling pathway has been shown to regulate proliferation and differentiation at a whole organ level in other systems. Importantly, at least in cell culture, the downstream effectors of the pathway, Yes-associated Protein (YAP) and Tafazzin (TAZ), can mediate mechanical signals to control proliferation and differentiation in a Hippo-independent fashion. Therefore, we hypothesize that Hippo signaling acts cell-autonomously in the incisor stem cells to determine their transition from proliferation to differentiation and YAP/TAZ additionally mediate mechanical cues to regulate stem cell behavior. Study design: By means of microarray and immunostaining, preliminary results show that Hippo components are expressed in the incisor stem cells. However, a detailed description of their spatial expression remains to be conducted and will be addressed in Aim 1 by performing in situ hybridization and immunostaining, as well as by constructing a YAP activity reporter mouse. In Aim 2, the proposed project will interrogate the cell-autonomous function of Hippo signaling specifically in the incisor stem cell population by removing MST1/2 or SAV, key components of the pathway, using Sox2-Cre. If a phenotype is observed, YAP will be similarly ablated in the mutant background to determine if it acts downstream of the pathway. Furthermore, the unique strength of this proposal lies in combining mouse genetics with biomechanical approaches to study how YAP/TAZ may relay mechanical signals to regulate stem cell behavior. In Aim 3, a tissue stiffness map of the mouse incisor will be constructed by using atomic force microscopy. These values will be used to cast hydrogels with different stiffness, on which freshly harvested wild type or YAP/TAZ mutant incisor stem cells will be cultured and investigated for their responses to mechanical signals. Health relatedness: As uncontrolled cell growth can lead to cancer and is therefore undesirable in stem cell-based therapies, successful completion of this multidisciplinary study will provide both genetic and biomechanical targets for the development of culturing strategies to derive and maintain dental stem cells that can be used to make replacement teeth that are clinically safe for treating patients with tooth loss.